Project Summary: Peroxisome proliferator-activated receptor (PPAR) coactivator-1 alpha (PGC-1) is a transcriptional coactivator that regulates diverse programs of energy metabolism in mammals by activating the expression of specific target genes in response to specific stimuli. Its activity is very tightly regulated in response to many signals such as energy demand and oxidative stress. Misregulation of PGC-1 is implicated in the etiology of many human diseases including obesity, diabetes, and neurologic disorders. The long-term goal of this research is to determine molecular mechanisms that regulate PGC-1 and develop therapies to manipulate those pathways in order to restore PGC-1 activity in PGC-1-deficient diseases. The specific aims of this proposal are to i) identify and characterize cellular mechanisms that regulate PGC-1 in response to oxidative stress and ii) to characterize the mechanism of a newly identified PGC-1 regulator, HectH9. Despite the plethora of information on PGC-1 regulation, the mechanism of PGC-1 activation by oxidative stress is not yet understood and until now HectH9 has not been identified as a PGC-1 regulator. One mode of PGC-1 regulation is through ubiquitylation. Students in the PIs lab discovered that the ubiquitin ligase HectH9 stabilizes PGC-1 rather than targeting it for proteolysis like canonical E3 ubiquitin ligases. Intriguingly, HectH9 regulation requires lysines in PGC-1 previously known to be acetylated. This proposal will test a model that predicts that HectH9 ubiquitylates PGC-1 lysines through alternatively-linked ubiquitin chains, thus antagonizing acetylation at these lysines and preventing other ubiquitin ligases from targeting PGC-1 for proteolysis. This proposal will test this model and determine the mechanism by which HectH9 stabilizes PGC-1 using siRNA knockdown and overexpression of HectH9 in conjunction with inhibition of the ubiquitin or acetylation pathways. The stability and transcriptional activity of PGC-1 will be evaluated. Furthermore, ubiquitylation assays will be used to determine the location of and branching pattern of the ubiquitin chains. HectH9 will also be evaluated for a role in the PGC-1 oxidative stress response. To discover other proteins involved in the PGC-1 antioxidant response, PGC-1 will be purified from cells exposed to oxidative stress and copurifying proteins identified by mass spectrometry. Four novel candidates have already been identified by this method. Three of these are antioxidant proteins suggesting a novel mechanism of PGC-1 activation in response to oxidative stress; oxidized forms of antioxidant proteins may stimulate PGC-1 after binding. This proposal will test the candidates already identified (and newly identified ones) by siRNA and overexpression experiments. The mechanism by which 1-3 promising candidates regulate PGC-1 and what physiologic signal stimulates them will be evaluated. This research is innovative as it will discover novel PGC-1 regulatory pathways and it proposes to test a model that suggests a new regulatory paradigm involving antagonism between ubiquitylation and acetylation. PUBLIC HEALTH RELEVANCE: Project Narrative Several forms of human diseases including diabetes, obesity, and neurological disorders have recently been discovered to result from a decrease in the beneficial human protein known as PGC-1. This proposal is designed to discover human proteins responsible for regulating PGC-1 function in human cells. Negative regulatory proteins will be excellent candidates against which to design inhibitory drugs or other therapies that consequently increase the activity of PGC-1 as treatment for these devastating diseases.